An Improved Method for Protein Identification that Incorporates Analysis of both MS and MS/MS Spectral Data. We have developed a method for protein identifications based on the combined analysis of MS and MS/MS spectral data collected from tryptic digests of proteins in gel bands using MALDI TOF-TOF instrumentation. The method uses theoretical peptide masses and measurement errors observed in the matched MS spectra to confirm protein identifications obtained from a first pass MS/MS database search. The method makes use of the mass accuracy of the MS1-level spectral data that have heretofore been ignored by most peptide database search engines. A probability model was developed to analyze the distribution of mass errors of peptide matches in the MS1 spectrum to provide a confidence level to the additional peptide matches. These additional matches are independent of the MS/MS database search identifications, and provide additional corroboration to identifications from MS/MS-based scores that are otherwise considered to be only of moderate quality. Straightforward and easily applicable to current proteomic analyses, this ProteinProcessor provides a robust and invaluable addition to current protein identification tools (3). A Mass Spectrometry Assay for Quantitation of N-Acetyltryptamine, N-Acetylserotonin and Melatonin (5-methoxy N-acetyltryptamine) in Plasma and Tissues. An MRM based assay to quantify N-acetyltryptamine and melatonin in plasma has been developed to study daily changes in tryptophan metabolites that may play a physiological role for actions mediated by the melatonin receptor. N-acetyltryptamine is a melatonin receptor mixed agonist/antagonist, and this assay has provided the first evidence for the presence of N-acetyltryptamine in plasma from human, rats, and rhesus monkeys. The liquid chromatography/tandem mass spectrometric method employs deuterated internal standards to quantitate N-acetyltryptamine and melatonin. N-acetyltryptamine was detected in daytime plasma from human volunteers, rhesus macaques and rats. Twenty-four hour studies of rhesus macaque plasma revealed that N-acetyltryptamine increases at night to concentrations that exceed those of melatonin. These findings establish the physiological presence of N-acetyltryptamine in circulation and support the hypothesis that this compound may play a significant physiological role as an endocrine or paracrine chonobiotic though actions mediated by the melatonin receptor. Ion Mobility Mass Spectrometry for Improved Detection of Complex Mixtures of Biomolecules. The Facility has installed the first commercial Ion Mobility Q-TOF LC/MS instrument (Agilent Technologies, Model 6560). The goal of implementing ion mobility spectrometry (IMS) prior to mass analysis is to add a dimension of separation to sample analysis that is orthogonal to both chromatography and mass spectrometry. Since the IMS operates on a millisecond time scale, the device offers the possibility of performing separations of complex mixtures at a much higher rate than possible with traditional LC methods. In addition, since IMS separations are associated with collision cross section (CCS) of ions (CCS is essentially a 'shape' parameter of ions in the gas phase), molecules of identical molecular weights can potentially be separated from one another on the basis of their CCS. This has implications for separations of isobaric steroids, lipids, peptides and proteins. IMS also offers the possibility of studying intermolecular complexes and their stoichiometry. One of the initial studies being undertaken is to investigate cyclodextrin-cholesterol complexes. Combined ion-mobility/mass spectrometry has also been used to analyze phospholipids. We investigated the incorporation of branched chain fatty acids into phosphatidylcholine (PC) in mice fed a diet supplemented with phytol (a C20 branched chain alcohol). Phytol is metabolized to phytanic acid that is then incorporated into phospholipids and triglycerides. Phospholipids extracted from muscle of control and phytol treated mice were analyzed by ion-mobility/mass spectrometry. To confirm the presence of phytanic acid, the ion-mobility of these species was compared with diphytanoyl PC standard and endogenous straight chain PC phospholipids. The muscle phosphatidylcholine species profiles were similar between mice treated with phytol and control diet, except that some additional species were detected in the phytol diet muscle. The two most abundant novel species (m/z 790.7 and 862.7) were tentatively identified as PC 20:0-16:0 and PC 20:0-22:6. The ion-mobility of these species were compared with diphytanoyl PC standard and endogenous straight chain PC phospholipids to demonstrate the separation of branch-chain phospholipids from those with straight chain fatty acids, and the ion-mobility of the novel PC species were consistent with incorporation of one phytanic acid. Identification of LECT2-associated Amyloidosis in Adrenal Tissue. We recently identified leukocyte cell-derived chemotaxin-2 (LECT2) as a component in the formation of amyloid plaques in adrenal tissue. Although adrenal tissue was positive for amyloid by Congo Red staining, specific immuno-staining for proteins commonly known to form amyloid plaques were all negative. Plaque proteins from disease tissue were extracted and separated by 1D SDS/PAGE. After digestion of the gel bands, serum amyloid P-component and leukocyte cell-derived chemotaxin-2 (LECT2) were identified as components of these plaques (5). Other reports have previously observed LECT2 in amyloid plaques in kidney, but this is the first observation of LECT2 amyloidosis in adrenal tissue. The high accumulation of LECT2 in adrenal amyloid plaques from this patient was confirmed by Western blots using a LECT2-specific antibody, which were positive for the disease tissue, while negative for comparable amounts of tissue from normal adrenal glands. Mass Spectrometric-Based Profiling of Urinary Steroids. Current approaches to the analysis of urinary steroids typically employ either immunoassay or mass spectrometry based technologies. Immunoassay-based methods often lack specificity due to cross-reactivity with other steroids, while targeted LC-MS/MS is limited to the analysis of pre-determined analytes. We have developed a new LC-MS/MS approach to urinary steroid profiling that enables us to detect the steroids that have truly changed in a patient cohort without knowing their identity beforehand (i.e., untargeted metabolomics of steroids). In addition, we have developed a product ion spectrum database of known steroids to improve our capability to identify novel steroids. These methods have been applied to a pilot project to investigate urinary steroids for patients diagnosed with polycystic ovarian syndrome (PCOS). Initially these studies detected elevated levels of an unknown compound consistent with an androgenic steroid in PCOS patients. We were then able to identify the unknown compound consistent with an androgenic steroid in PCOS patients. We were then able to identify the unknown as a mixture of androsterone-sulfate and etiocholanolone-sulfate. We have also developed a reversed phase LC-MRM method to quantitate alpha and beta p-Diol levels in urine. These compounds are intermediates in the backdoor pathway for androgen synthesis. This assay has been applied to studies of patients with Congenital Adrenal Hyperplasia (CAH). Both alpha and beta p-Diol levels in urine were found to correlate with serum androgen levels, suggesting that these compounds could be novel biomarkers to monitor CAH disease control by glucocorticoid treatment (2).